Ligase IV has been shown to play critical roles in Non-Homologous End Joining (NHEJ), V(D)J recombination and development of the immune system, stem cell exhaustion, ageing and neural growth and development, as exemplified by the phenotype of patients with hypomorphic mutations in Ligase IV. Numerous proteins have been shown to modulate Ligase IV activity. Among them, XRCC4 and Cernunnos/XLF are perhaps the best/most conclusively characterized. Both of these factors, like Ligase IV, are part of the core-NHEJ machinery. Human mutations in XRCC4 have not been described yet, but interestingly, Cernunnos/XLF mutations in humans result in growth defects, microcephaly and immunodeficiency, similar to what has been observed for Ligase IV mutations. Our recent findings have identified Artemis, a factor also linked to immunodeficiency in humans, as a protein that directly interacts with Ligase IV, in addition we have observed regulation of XRCC4 by Ligase IV. In this project we propose to analyze the functional relevance of these novel Ligase IV/Artemis complex in genomic stability and V(D)J recombination. The mechanism by which Ligase IV regulates XRCC4 function, and the composition and function of endogenous Ligase IV complexes in lymphocytes will also be investigated. Our preliminary work with Ligase IV recombinant protein and protein fragments has resulted in crystallization of a Ligase IV fragment containing the Ligase IV DNA binding domain. Experiments are proposed to solve the structure of Ligase IV and/or Ligase IV in complex with Artemis. All these are challenging yet very relevant experiments for which we show significant progresses. Furthermore, the impact of mutations identified in patients with LIG4 syndrome on regulation of its function by Artemis and on how these mutant Ligase IV proteins affect XRCC4 function will also be investigated. Information gained from the proposed studies will contribute to a molecular and structural understanding of NHEJ, V(D)J recombination and human diseases linked to defects in both of these processes. PUBLIC HEALTH RELEVANCE: Despite important progress in our understanding of Ligase IV function, its structure remains largely uncharacterized and our preliminary data suggest novel mechanisms of regulation that remain to be unraveled. This project proposes to investigate our preliminary finding on novel Ligase IV interactions and functions by using in vivo and in vitro approaches;we also propose studies to solve the structure of Ligase IV and Ligase IV complexes. By increasing our knowledge of Ligase IV structure, function and regulation, in V(D)J recombination and DNA repair, the proposed work will contribute to our understanding of human disease, specifically, Ligase IV and Artemis mediated, immunodeficiency and cancer and will facilitate the path to find better treatments for this debilitating diseases.